Inventory of Supplemental Information for Melkman-Zehavi et al.

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1 Inventory of Supplemental Information for Melkman-Zehavi et al. Supplementary methods for Melkman et al. Supplementary Figures Figure S1, related to Figure 2 Islet size and total beta cell mass of mutant animals is not significantly different from that of control mice. Analysis of pancreatic tissue that was isolated from agematched animals at 3 weeks post Tamoxifen treatment. (A) The mean (± SEM) beta cell mass of control (n = 2) and mutant animal (n = 3) tissues was calculated by morphometry. (B) Comparable distribution of islet size (displayed as area of insulin staining) between control and mutant animals. (C) Comparable and low apoptosis frequency detected in three control and three mutant animals (>20 islets per animal). Figure S2, related to Figure 2 Mutant beta cells maintain normal PC1/3 expression. Immunostaining of GFP expression (A,D) and PC1/3 (B,E) shows their colocalization in the merged picture (yellow, C,F) in both control (A-C) and mutant (D-F) animals. Figure S3 related to Figure 4 Mutant beta cells maintain expression of beta cell markers. Quantification of individual beta cells co-expressing insulin and transcription factors relevant to insulin transcription: Pax6, NKX6.1 and MAFA (control) and beta cells that underwent recombination, co-expressing GFP and the same set of transcription factors. Three animals in each group; >27 islets; >1300 individual beta cells per group. Figure S4 Expression array analysis of mirna expressed in adult mouse islets. A vertical histogram listing mirna expressed in isolated islets, listed by their relative fluorescence intensity after hybridization to a home-printed microarray (Ambion). Note that the design of this array did not contain features for mir-375.

2 Supplementary methods for Melkman et al.: Primers for PCR genotyping. Gene Forward primer Reverse primer Dicer1 CCTGACAGTGACGGTCCAAAG CATGACTCTTCAACTCAAACT Cre TGCCACGACCAAGTGACAGC CCAGGTTACGGATATAGTTCATG GFP CCTACGGCGTGCAGTGCTTCAGC CGGCGAGCTGCACGCTGCGTCCTC Primers for quantitative real-time PCR of mrnas. Gene Forward primer Reverse primer Dicer1 CACGCCTCCTACCACTACAACA CCTGGAGAATGCTGCCGTGGGT insulin1 CCTGTTGGTGCACTTCCTAC TGCAGTAGTTCTCCAGCTGG insulin2 CGTGGCTTCTTCTACACACCC AGCTCCAGTTGTGCCACTTGT Pdx1 TTCCCGAATGGAACCGAGC GTAGGCAGTACGGGTCCTCT Nkx2.2 CCGGGCGGAGAAAGGTATG CTGTAGGCGGAAAAGGGGA Nkx6.1 TCAGTCAAGGTCTGGTTCC CGATTTGTGCTTTTTCAGCA MafA AGGAGGAGGTCATCCGACTG CTTCTCGCTCTCCAGAATGTG NeouroD1 GACCCAGAAACTGTCTAAAATAGAGACA AAGGAGACCAGATCAGGGCTTT Stx3 GAAGGCACGGGATGAAACTAA GGACAGTCCAATAATCAACGCTA Hes1 GTCTAAGCCAACTGAAAACACTGATT TGCCTTCTCTAGCTTGGAATGC Insm1 CGGCCACCTTCTACAGCTC GGAGGATCACCTGTCTATTCTCA Crem GCTGAGGCTGATGAAAAACA GCCACACGATTTTCAAGACA Sox6 GACAGCGTTCTGTCATCTCAGCAA CGTTCCGGGGTTCCAAAAGTAACA Tle4 TTTACAGGCTCAATACCACAGTC TGCACAGATAGCATTTAGTCGTT Bhlhe22 GGGGAGAGGGAGGTTTAGTG CCCTTTCATCACTTGCCAAT HPRT CTGGTTAAGCAGTACAGCCCCAAA TGGCCTGTATCCAACACTTCGAGA GAPDH TGGCAAAGTGGAGATTGTTGCC AAGATGGTGATGGGCTTCCCG Primary antibodies Antibody guinea pig anti-insulin rabbit anti-glucagon rabbit anti-somatostatin goat anti-gfp Concentration and manufacturer 1:200; DAKO 1:200; DAKO 1:200; Zymed 1:100; Abcam

3 rabbit anti-gfp rabbit anti-pdx1 Rabbit anti-ngn3 mouse anti-nkx6.1 Rabbit anti-mafa Rabbit anti-synaptophysin Rabbit anti-pax6 Goat anti-ghrelin rabbit anti-activated caspase-3 rabbit anti-prohormone convertase 1/3 1:250; Invitrogen 1: 5000 Beta Cell Biology Consortium 1:300; Santa cruz 1:100; hybridoma bank 1:100, Bethyl 1:100, DAKO 1:300; Chemicon 1:50; Santa cruz 1:50, cell signaling 1:100; Chemicon Secondary antibodies conjugated to CY2, CY3, or CY5 were all from Jackson Immunoresearch Laboratories (1:100). Histomorphometry To determine beta cell mass, consecutive paraffin sections 5 μm in thickness and 75 μm apart spanning the entire pancreas (approximately 9 sections/ pancreas) were stained for insulin and counterstained using Harris hematoxylin (Sigma). Digital images of sections were obtained at a low magnification ( 40) and stitched using NIS-Elements software. The fraction of beta cells was determined by measuring the area of insulin immuno-reactivity divided by the area of the whole pancreas, determined by counter-staining with hematoxylin. The mass of beta cells was calculated as the product of pancreas weight and the fraction of tissue covered by beta cells. Anti-miRNA Oligonucleotide (AMO) synthesis and purification. AMOs were designed as reverse complements to the targeted mirnas and were made with 2 -O-Methyl RNA (2 OMe) bases with phosphodiester linkages using standard phosphoramidite chemistry at Integrated DNA Technologies (Coralville, IA, USA). Each AMO had a non-nucleotide chemical modifier, called ZEN inserted between the last and next to last base on both the 5 - and 3 -ends.this modifier is a napthylazo group which non-specifically increases binding affinity (T m ) and also renders the

4 AMO resistant to exonuclease degradation (K.A. Lennox and M.A. Behlke, manuscript in preparation). Each AMO also had a 3 -cholesterol-teg modifier (Glen Research, Sterling, VA, USA). The AMOs were purified using reversed-phase high performance liquid chromatography (RP-HPLC). Electrospray-ionization liquid chromatography mass spectroscopy (ESI-LCMS) of the oligonucleotides was conducted using an Oligo HTCS system (Novatia, Princeton, NJ), which consisted of ThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processing software and Paradigm MS4 HPLC (MichromBioResources, Auburn, CA). Experimental molar masses for all single strand oligomers were within 1.5 g/mol of expected mass. Oligonucleotides were prepared as sodium salts. The NC1 AMO is a non-targeting control oligonucleotide that is not complementary to any entry in mirbase. A list of all AMOs employed in this study are listed in the table below. Name Sequence (5 3 ) NC1 AMO Let-7cAMO Let-7fAMO mmu-mir-7aamo mmu-mir-9 AMO mmu-mir-24amo mmu-mir-26aamo mmu-mir-27bamo mmu-mir-30aamo mmu-mir-141amo mmu-mir-148aamo mmu-mir-182amo mmu-mir-200camo mmu-mir-375amo G z CGUAUUAUAGCCGAUUAACG z A-Chol A z ACCAUACAACCUACUACCUC z A-Chol A z ACUAUACAAUCUACUACCUC z A-Chol A z CAACAAAAUCACUAGUCUUCC z A-Chol U z CAUACAGCUAGAUAACCAAAG z A-Chol C z UGUUCCUGCUGAACUGAGCC z A-Chol A z GCCUAUCCUGGAUUACUUGA z A-Chol G z CAGAACUUAGCCACUGUGA z A-Chol C z UUCCAGUCGAGGAUGUUUAC z A-Chol C z CAUCUUUACCAGACAGUGUU z A-Chol A z CAAAGUUCUGUAGUGCACUG z A-Chol C z GGUGUGAGUUCUACCAUUGCCAA z A-Chol U z CCAUCAUUACCCGGCAGUAUU z A-Chol U z CACGCGAGCCGAACGAACAA z A-Chol

5 A 3 control mutant Beta cell mass (mg) 2 1 B % of total events >50001 µm µm µm µm µm µm 2 0 control mutant C Caspase positive cells (%) control mutant Supp. figure 1

6 GFP PC1/3 Merge A B C mutant Control D E F Supp. figure 2

7 120 insulin+ cells GFP+ cells ** cells coexpressing the markers (%) Nkx6.1 Pax6 MafA Supp Figure 3

8 Intensity Intensity Supp. figure 4